In vivo production and delivery of erythropoietin for gene therapy

ABSTRACT

The present invention relates to transfected primary and secondary somatic cells of vertebrate origin, particularly mammalian origin, transfected with exogenous genetic material (DNA) which encodes erythropoietin or an insulinotropin [e.g., derivatives of glucagon-like peptide 1(GLP-1)], methods by which primary and secondary cells are transfected to include exogenous genetic material encoding erythropoietin or an insulinotropin, methods of producing clonal cell strains or heterogenous cell strains which express eruthropoietin or an insulinotropin, methods of gene therapy in which the transfected primary or secondary cells are used, the methods of producing antibodies using the transfected primary or secondary cells.

RELATED APPLICATIONS

This application is a division of application Ser. No. 08/334,455 filedNov. 4, 1994, which is a File Wrapper Continuation of Ser. No.07/911,533 filed Jul. 10, 1992 now abandoned which is aContinuation-in-Part of Ser. No. 07/787,840 filed Nov. 5, 1991, nowabandoned and also of Ser. No. 07/789,188, filed Nov. 5, 1991, nowabandoned.

BACKGROUND OF THE INVENTION

A variety of congenital, acquired, or induced syndromes are associatedwith insufficient numbers of erythrocytes (red blood cells or RBCs). Theclinical consequence of such syndromes, collectively known as theanemias, is a decreased oxygen-carrying potential of the blood,resulting in fatigue, weakness, and failure-to-thrive. Erythropoietin(EPO), a glycoprotien of molecular mass 34,000 daltons, is synthesizedand released into the systemic circulation in response to reduced oxygentension in the blood. EPO, primarily synthesized in the kidney and, to alesser extent, in the liver, acts on erythroid precursor cells [ColonyForming Units-Erythroid (CFU-E) and Burst-Forming Units-Erythroid(BFU-E)] to promote differentiation into reticulocytes, and ultimately,mature erythrocytes.

The kidney is the major site of EPO production and, thus, renal failureor nephrectomy can lead to decreased EPO synthesis, reduced RBC numbers,and, ultimately, severe anemia as observed in predialysis and dialysispatients. Subnormal RBC counts may also result from the toxic effects ofchemotherapeutic agents or azidothymidine (AZT) (used in the treatmentof cancers and AIDS, respectively) on erythroid precursor cells. Inaddition, a variety of acquired and congenital syndromes, such asaplastic anemia, myeloproliferative syndrome, malignant lymphomas,multiple myeloma, neonatal prematurity, sickle-cell anemia, porphyriacutanea tarda, and Gaucher's disease include anemia as one clinicalmanifestation of the syndrome.

Purified human EPO or recombinant human EPO may be administered topatients in order to alleviate anemia by increasing erythrocyteproduction. Typically, the protein is administered by regularintravenous injections. The administration of EPO by injection is animperfect treatment. Normal individuals maintain a relatively constantlevel of EPO, which is in the range of 6-30 mU/ml, depending on theassay used. After typical treatment regimens, serum EPO levels may reach3,000-5,000 mU/Ml following a single injection, with levels falling overtime as the protein is cleared from the blood.

If a relatively constant level of EPO is to be provided in the blood(i.e., to mimic the normal physiology of the protein), a delivery systemthat is capable of releasing a continuous, precisely dosed quantity ofEPO into the blood is necessary.

SUMMARY OF THE INVENTION

The present invention relates to transfected primary and secondarysomatic cells of vertebrate origin, particularly mammalian origin,transfected with exogenous genetic material (DNA or RNA) which encodes aclinically useful product, such as erythropoietin (EPO) orinsulinotropin [e.g. derivatives of glucagon-like peptide 1 (GLP-1) suchas GLP(7-37), GLP(7-36), GLP-1(7-35) and GLP-1(7-34) as well as theircarboxy-terminal amidated derivatives produced by in vivo amidatingenzymes and derivatives which have amino acid alterations or otheralterations which result in substantially the same biological activityor stability in the blood as that of a truncated GLP-1 or enhancedbiological activity or stability], methods by which primary andsecondary cells are transfected to include exogenous genetic materialencoding EPO or insulinotropin, methods of producing clonal cell strainsor heterogenous cell strains which express exogenous genetic materialencoding EPO or insulinotropin, a method of providing EPO orinsulinotropin in physiologically useful quantitites to an individual inneed thereof, through the use of transfected cells of the presentinvention or by direct injection of DNA encoding EPO into an individual;and methods of producing antibodies against the encoded product usingthe transfected primary or secondary cells. Transfected cells containingEPO-encoding exogenous genetic material express EPO and, thus, areuseful for preventing or treating conditions in which EPO productionand/or utilization are inadequate or compromised, such as in anycondition or disease in which there is anemia. Similarly, transfectedcells containing insulinotropin-encoding exogenous genetic materialexpress insulinotropin and, thus, are useful for treating individuals inwhom insulin secretion, sensitivity or function is compromised (e.g.,individuals with insulin-dependent or non-insulin dependent diabetes).

The present invention includes primary and secondary somatic cells, suchas fibroblasts, keratinocytes, epithelial cells, endothelial cells,glial cells, neural cells formed elements of the blood, muscle cells,other somatic cells which can be cultured and somatic cell precursors,which have been transfected with exogenous DNA encoding EPO or exogenousDNA encoding insulinotropin. The exogenous DNA is stably integrated intothe cell genome or is expressed in the cells episomally. The exogenousDNA encoding EPO is introduced into cells operatively linked withadditional DNA sequences sufficient for expression of EPO in transfectedcells. The exogenous DNA encoding EPO is preferably DNA encoding humanEPO but, in some instances, can be DNA encoding mammalian EPO ofnon-human origin. EPO produced by the cells is secreted from the cellsand, thus, made available for preventing or treating a condition ordisease (e.g., anemia) in which EPO production and/or utilization isless than normal or inadequate for maintaining a suitable level of RBCs.Cells produced by the present method can be introduced into an animal,such as a human, in need of EPO and EPO produced in the cells issecreted into the systemic circulation. As a result, EPO is madeavailable for prevention or treatment of a condition in which EPOproduction and/or utilization is less than normal or inadequate tomaintain a suitable level of RBCs in the individual. Similarly,exogenous DNA encoding insulinotropin is introduced into cellsoperatively linked with additional DNA sequences sufficient forexpression of insulinotropin in transfected cells. The encodedinsulinotropin is made available to prevent or treat a condition inwhich insulin production or function is compromised or glucagon releasefrom the pancreas is to be inhibited.

Primary and secondary cells transfected by the subject method can beseen to fall into three types or categories: 1) cells which do not, asobtained, produce and/or secrete the encoded protein (e.g., EPO,insulinotropin; 2) cells which produce and/or secrete the encodedprotein (e.g., EPO, insulinotropin) but in lower quantities than normal(in quantities less than the physiologically normal lower level) or indefective form, and 3) cells which make the encoded protein (e.g., EPOor insulinotropin) at physiologically normal levels, but are to beaugmented or enhanced in their production and/or secretion of theencoded protein.

Exogenous DNA encoding EPO is introduced into primary or secondary cellsby a variety of techniques. For example, a construct which includesexogenous DNA encoding EPO and additional DNA sequences necessary forexpression of EPO in recipient cells is introduced into primary orsecondary cells by electroporation, microinjection, or other means(e.g., calcium phosphate precipitation, modified calcium phosphateprecipitation, polybrene precipitation, microprojectile bombardment,liposome fusion, receptor-mediated DNA delivery). Alternatively, avector, such as retroviral vector, which includes exogenous DNA encodingEPO can be used, and cells can be genetically modified as a result ofinfection with the vector. Similarly, exogenous DNA encodinginsulinotropin is introduced into primary of secondary cells using oneof a variety of methods.

In addition to exogenous DNA encoding EPO or insulinotropin, transfectedprimary and secondary cells may optionally contain DNA encoding aselectable marker, which is expressed and confers upon recipient cells aselectable phenotype, such as antibiotic resistance, resistance to acytotoxic agent, nutritional prototrophy or expression of a surfaceprotein. Its presence makes it possible to identify and select cellscontaining the exogenous DNA. A variety of selectable marker genes canbe used, such as neo, gpt, dhfr, ada, pac, hyg, mdr and hisD.

Transfected cells of the present invention are useful, as populations oftransfected primary cells, transfected clonal cell strains, transfectedheterogenous cell strains, and as cell mixtures in which at least onerepresentative cell of one of the three preceding categories oftransfected cells is present, as a delivery system for treating anindividual with a condition or disease which responds to delivery of EPO(e.g. anemia) or for preventing the development of such a condition ordisease. In the method of the present invention of providing EPO,transfected primary cells, clonal cell strains, or heterogenous cellstrains, are administered to an individual in need of EPO, in sufficientquantity and by an appropriate route, to deliver EPO to the systemiccirculation at a physiologically relevant level. In a similar manner,transfected cells of the present invention providing insulinotropin areuseful as populations of transfected primary cells, transfected clonalcell strains, transfected heterogenous cell strains, and as cellmixtures, as a delivery system for treating an individual in whominsulin production, secretion or function is comprised or for inhibiting(totally or partially) glucagon secretion from the pancreas. Aphysiologically relevant level is one which either approximates thelevel at which the product is normally produced in the body or resultsin improvement of an abnormal or undesirable condition.

Clonal cell strains of transfected secondary cells (referred to astransfected clonal cell strains) expressing exogenous DNA encoding EPO(and, optionally, including a selectable marker gene) are produced bythe method of the present invention. The present method includes thesteps of: 1) providing a population of primary cells, obtained from theindividual to whom the transfected primary cells will be administered orfrom another source; 2) introducing into the primary cells or intosecondary cells derived from primary cells a DNA construct whichincludes exogenous DNA encoding EPO and additional DNA sequencesnecessary for expression of EPO, thus producing transfected primary orsecondary cells; 3) maintaining transfected primary or secondary cellsunder conditions appropriate for their propagation; 4) identifying atransfected primary or secondary cell; and 5) producing a colony fromthe transfected primary or secondary cell identified in (4) bymaintaining it under appropriate culture conditions and for sufficienttime for its propagation, thereby producing a cell strain derived fromthe (founder) cell identified in (4). In one embodiment of the method,exogenous DNA encoding EPO is introduced into genomic DNA by homologousrecombination between DNA sequences present in the DNA construct used totransfect the recipient cells and the recipient cell's genomic DNA.Clonal cell strains of transfected secondary cells expressing exogenousDNA encoding insulinotropin (and, optionally, including a selectablemarker gene) are also produced by the present method.

In one embodiment of the present method of producing a clonal populationof transfected secondary cells, a cell suspension containing primary orsecondary cells is combined with exogenous DNA encoding EPO and DNAencoding a selectable marker, such as the bacterial neo gene. The twoDNA sequences are present on the same DNA construct or on two separateDNA constructs. The resulting combination is subjected toelectroporation, generally at 250-300 volts with a capacitance of 960μFarads and an appropriate time constant (e.g., 14 to 20 msec) for cellsto take up the DNA construct. In an alternative embodiment,microinjection is used to introduce the DNA construct containingEPO-encoding DNA into primary or secondary cells. In either embodiment,introduction of the exogenous DNA results in production of transfectedprimary or secondary cells. Using the same approach, electroporation ormicroinjection is used to produce a clonal population of transfectedsecondary cells containing exogenous DNA encoding insulinotropin alone,or insulinotropin and a selectable marker.

In the method of producing heterogenous cell strains of the presentinvention, the same steps are carried out as described for production ofa clonal cell strain, except that a single transfected primary orsecondary cell is not isolated and used as the founder cell. Instead,two or more transfected primary or secondary cells are cultured toproduce a heterogenous cell strain.

The subject invention also relates to a method of producing antibodiesspecific for EPO. In the method, transfected primary or secondary cellsexpressing EPO are introduced into an animal recipient (e.g., rabbit,mouse, pig, dog, cat, goat, guinea pig, sheep, non-human primate). Theanimal recipient produces antibodies against the EPO expressed, whichmay be th entire EPO protein antigen or a peptide encoded by a fragmentof the intact EPO gene. Polyclonal sera is obtained from the animals. Itis also possible to produce monoclonal antibodies through the use oftransfected primary or secondary cells. Splenocytes are removed from ananimal recipient of transfected primary or secondary cells expressingEPO. The splenocytes are fused with myeloma cells, using known methods,such as that of Koprowski et al. (U.S. Pat. No. 4,172,124) or Kohler etal., (Nature 256: 495-497 (1975)) to produce hybridoma cells whichproduce the desired anti-EPO monoclonal antibody. The polyclonalantisera and monoclonal antibodies produced can be used for the samepurposes (e.g., diagnostic, preventive, or therapeutic purposes) asantibodies produced by other methods. Similarly, antibodies specific forinsulinotropin can be produced by the method of the present invention.

The present invention is particularly advantageous in treating anemiaand other conditions in which EPO production, utilization or both iscompromised in that it: 1) makes it possible for one gene therapytreatment, when necessary, to last a patient's lifetime; 2) allowsprecise dosing (the patient's cells continuously determine and deliverthe optimal dose of EPO based on physiologic demands, and the stablytransfected cell strains can be characterized extensively in vitro priorto implantation, leading to accurate predictions of long term functionin vivo); 3) is simple to apply in treating patients; 4) eliminatesissues concerning patient compliance (periodic administration of EPO isno longer necessary); and 5) reduces treatment costs (since thetherapeutic protein is synthesized by the patient's own cells,investment in costly protein production and purification facilities isunnecessary).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of plasmid pXEPO1. The solid blackarc represents the pUC12 backbone and the arrow denotes the direction oftranscription of the ampicillin resistance gene. The stippled arcrepresents the mouse metallothionein promoter (pmMT1). The unfilled arcinterrupted by black boxes represents the human erythropoietin EPO gene(the black boxes denote exons and the arrow indicates the direction hEPOtranscription). The relative positions of restriction endonucleaserecognition sites are indicated.

FIG. 2 is a schematic representation of plasmid pcDNEO. This plasmid hasthe neo gene from plasmid pSV2neo (a BamHI-BgIII fragment) inserted intothe BamHI site of plasmid pcD; the amp and pBR322ori sequences are frompBR322; and polyA, 19S splice junction, and early promoter sequences arefrom SV40.

FIG. 3 is a schematic representation of plasmid pXGH301. This plasmidcontains both the human growth hormone (hGH) and neo resistance genes.Arrows indicate the directions of transcription of the various genes.The positions of restriction endonuclease recognition sites, the mousemetallothionein promoter (pMMT1), the amp resistance gene, and the SV40early promoter (pSV40 early) are indicated.

FIG. 4 is a schematic representation of plasmid pE3neoEPO. The positionsof the human erythropoietin gene and the neo and amp resistance genesare indicated. Arrows indicate the directions of the transcription ofthe various genes. pmMT1 denotes the mouse metallothionein promoter(driving hEPO expression) and pTK denotes the Herpes Simplex Virusthymidine kinase promoter (driving neo expression). The dotted regionsof the map mark the positions of human HGPRT sequences. The relativepositions of restriction endonuclease recognition sites are indicated.

FIG. 5A shows results of Western blot analysis of hEPO secreted bynormal human fibroblasts cotransfected with pXEPO1 and pcDNEO. The leftpanel shows the Western analysis and the right panel shows a photographof the Coomassie blue stained gel. Lanes C,E, and M signify Controlsample (supernatant from untransfected human fibroblasts), Experimentalsample (supernatant from a clonal strain of human fibroblasts expressinghEPO), and marker lanes, respectively.

FIG. 5B shows results of Western blot analysis of hEPO secreted bynormal human fibroblasts cotransfected with pXEPO1 and pcDNEO.Supernatant from a clonal strain of human fibroblasts expressing hEPO(lane 1) was further analyzed for glycosylation by treatment withendoglycosidase-F (lane 2), neuraminidase (lane 3), and O-glycanase(lane 4).

FIG. 6A shows results of an assay to detect hEPO in the serum of miceimplanted with transfected rabbit fibroblasts expressing hEPO.

FIG. 6B shows hematocrit (HCT) levels in control mice and mice implantedwith transfected rabbit fibroblasts expressing hEPO.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of genetically engineered cellsto deliver a clinically useful or otherwise desirable substance to anindividual in whom production of the substance is desired (e.g., toprevent or treat a disease or condition in which the product is producedor functions at an acceptable level). In particular, it relates to theuse of genetically engineered cells to deliver EPO to the systemiccirculation of an individual in need of EPO, resulting in an increase inmature red blood cell numbers, an increase in the oxygen-carryingpotential of the blood, and an alleviation of the symptoms of anemia.The present invention provides a means of delivering EPO atphysiologically relevant levels and on a continuous basis to anindividual. It further particularly relates to the use of geneticallyengineered cells to deliver insulinotropin to an individual in need ofinsulinotropin to stimulate insulin release, to increase insulinsensitivity in peripheral tissues, or to inhibit glucagon secretion fromthe pancreas.

As used herein, the term primary cell includes cells present in asuspension of cells isolated from a vertebrate tissue source (prior totheir being plated, i.e., attached to a tissue culture substrate such asa dish or flask), cells present in an explant derived from tissue, bothof the previous types of cells plated for the first time, and cellsuspensions derived from these plated cells. The term secondary cell orcell strain refers to cells at all subsequent steps in culturing. Thatis, the first time a plated primary cell is removed from the culturesubstrate and replated (passaged), it is referred to herein as asecondary cell, as are all cells in subsequent passages. Secondary cellsare cell strains which consist of secondary cells which have beenpassaged one or more times. A cell strain consists of secondary cellsthat: 1) have been passaged one or more times; 2) exhibit a finitenumber of mean population doublings in culture; 3) exhibit theproperties of contact-inhibited, anchorage dependent growth(anchorage-dependence does not apply to cells that are propagated insuspension culture); and 4) are not immortalized. A "clonal cell strain"is defined as a cell strain that is derived from a single founder cell.A "heterogenous cell strain" is defined as a cell strain that is derivedfrom two or more founder cells.

As described herein, primary or secondary cells of vertebrate,particularly mammalian, origin have been transfected with exogenous DNAencoding EPO and shown to produce the encoded EPO reproducibly, both invitro and in vivo, over extended periods of time. In addition, thetransfected primary and secondary cells have been shown to express EPOin vivo at physiologically relevant levels. The EPO expressed has beenshown to have the glycosylation pattern typical of EPO purified fromhuman urine or recombinant human EPO. This demonstration is in sharpcontrast to what one of skill in the art would predict, since, forexample, even experts in the field see the finite life span of normalsomatic cells and the inability to isolate or grow the relevanttransplantable cells as precluding their use for gene therapy unless thecells are genetically modified using retroviruses (Miller, A. D., Blood,76:271-278 (1990)). However, the transplantation of retrovirally treatedfibroblasts has been shown to provide, at best, only transient metabolicimprovements, and is seen to have serious limitations as a therapeuticsystem. In addition, until Applicants' work, this had not been done forEPO. Normal (non immortal) fibroblasts are characterized as being "muchmore difficult to transfect than continuous cell lines by using calciumphosphate precipitation techniques." (Miller, A. D., Blood, 76:271-278(1990)). Furthermore, in considering non-retroviral techniques for genetherapy, it is typical of experts in the field to believe " . . . theefficiency of gene delivery is dismal . . . A physician would have toobtain an impossible number of cells for patients to guarantee theappropriate alteration of the millions required for therapy." (Verma, I.M. Scient. Amer., November 1990, pages 68-84).

Surprisingly, Applicants have been able to produce transfected primaryand secondary cells which include exogenous DNA encoding EPO and expressthe exogenous DNA.

The transfected primary or secondary cells may also include DNA encodinga selectable marker which confers a selectable phenotype upon them,facilitating their identification and isolation. Applicants have alsodeveloped methods for producing transfected primary or secondary cellswhich stably express exogenous DNA encoding EPO, clonal cell strains andheterogenous cell strains of such transfected cells, methods ofproducing the clonal and heterogenous cell strains, and methods of usingtransfected cells expressing EPO to deliver the encoded product to anindividual mammal at physiologically relevant levels. The constructs andmethods are useful, for example, for treating an individual (human)whose EPO production and/or function is in need of being increased orenhanced [e.g., is comprised or less than normal, or normal but theindividual would benefit from enhancement, at least temporarily, of redblood cell production (e.g., during predialysis or dialysis therapy,during treatment of AIDS with AZT, after surgery, or duringchemotherapy)].

As also described herein, it is possible to transfect primary orsecondary cells of vertebrate, particularly mammalian, origin withexogenous DNA encoding insulinotropin and to use them to provideinsulinotropin to an individual in whom insulin production, functionand/or sensitivity is compromised.

Transfected Cells

Primary and secondary cells to be transfected in order to produce EPO orinsulinotropin can be obtained from a variety of tissues and include allcell types which can be maintained and propagated in culture. Forexample, primary and secondary cells which can be transfected by thepresent method include fibroblasts, keratinocytes, epithelial cells(e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells, othersomatic cells which can be cultured, and precursors of these somaticcell types. Primary cells are preferably obtained from the individual towhom the transfected primary or secondary cells are administered.However, primary cells may be obtained from a donor (other than therecipient) of the same species or another species (e.g., mouse, rat,rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Transfected primary and secondary cells can be produced, with or withoutphenotypic selection, as described herein, and shown to expressexogenous DNA encoding EPO or exogenous DNA encoding insulinotropin.

Exogenous DNA

Exogenous DNA incorporated into primary or secondary cells by thepresent method is DNA encoding the desired product (e.g., EPO orinsulinotropin), a functional or active portion, or a functionalequivalent of EPO or insulinotropin (a protein which has a differentamino acid sequence from that of EPO but has substantially the samebiological function as EPO, or a protein which has a different aminoacid sequence from that of GLP-1 related peptides but functionsbiologically as insulinotropin). The DNA can be obtained from a sourcein which it occurs in nature or can be produced, using geneticengineering techniques or synthetic processes. The DNA encoding EPO orinsulinotropin will generally be DNA encoding the human product (i.e.,human EPO or human insulinotropin). In some cases, however, the DNA canbe DNA encoding EPO or insulinotropin of non-human origin (i.e., DNAobtained from a non-human source or DNA, produced recombinantly or bysynthetic methods, which encodes a non-human EPO or insulinotropin).

The DNA transfected into primary or secondary cells can encode EPO aloneor EPO and another product, such as a selectable marker to facilitateselection and identification of transfected cells. Alternatively, thetransfected DNA can encode insulinotropin alone or insulinotropin andanother product, such as a selectable marker. After transfection intoprimary or secondary cells, the exogenous DNA is stably incorporatedinto the recipient cell's genome (along with the additional sequencespresent in the DNA construct used), from which it is expressed orotherwise function. Alternatively, the exogeneous DNA may existepisomally within the transfected primary or secondary cells. DNAencoding the desired product can be introduced into cells under thecontrol of an inducible promoter, with the result that cells produced oras introduced into an individual do not express the product but can beinduced to do so (i.e., production is induced after the transfectedcells are produced but before implantation or after implantation). DNAencoding the desired product can, of course, be introduced into cells insuch a manner that it is expressed upon introduction (i.e., withoutinduction).

Selectable Markers

A variety of selectable markers can be incorporated into primary orsecondary cells. For example, a selectable marker which confers aselectable phenotype such as drug resistance, nutritional auxotrophy,resistance to a cytotoxic agent or expression of a surface protein, canbe used. Selectable marker genes which can be used include neo, gpt,dhfr, ada, pac, hyg and hisD. The selectable phenotype conferred makesit possible to identify and isolate recipient primary or secondarycells.

DNA Constructs

DNA constructs, which include exogenous DNA encoding the desired product(e.g., EPO, insulinotropin) and, optionally, DNA encoding a selectablemarker, along with additional sequences necessary for expression of theexogenous DNA in recipient primary or secondary cells, are used totransfect primary or secondary cells in which the protein (e.g., EPO,insulinotropin) is to be produced. Alternatively, infectious vectors,such as retroviral, herpes, adenovirus, adenovirus-associated, mumps andpoliovirus vectors, can be used for this purpose.

A DNA construct which includes the exogenous DNA encoding EPO andadditional sequences, such as sequences necessary for expression of EPO,can be used (e.g., plasmid pXEPO1; see FIG. 1). A DNA construct caninclude an inducible promoter which controls expression of the exogenousDNA, making inducible expression possible. Optionally, the DNA constructmay include a bacterial origin of replication and bacterial antibioticresistance markers, which allow for large-scale plasmid propagation inbacteria. A DNA construct which includes DNA encoding a selectablemarker, along with additional sequences, such as a promoter,polyadenylation site, and splice junctions, can be used to confer aselectable phenotype upon transfected primary or secondary cells (e.g.,plasmid pcDNEO). The two DNA constructs are cotransfected into primaryor secondary cells, using methods described herein. Alternatively, oneDNA construct which includes exogenous DNA encoding EPO, a selectablemarker gene and additional sequences (e.g.,), those necessary forexpression of the exogenous DNA and for expression of the selectablemarker gene) can be used. Such a DNA construct (pE3neoEPO) is describedin FIG. 4; it includes the EPO gene and the neo gene. Similarconstructs, which include exogenous DNA encoding insulinotropin andadditional sequences (e.g., sequences necessary for insulinotropinexpression) can be produced (e.g., plasmid pXGLP1; see FIG. 11). Theconstructs can also include DNA encoding a selectable marker, as well asother sequences, such as a promoter, a polyadenylation site, and splicejunctions.

In those instances in which DNA is injected directly into an individual,such as by injection intomusceles, the DNA construct includes theexogenous DNA and regulatory sequences necessary and sufficient forexpression of the encoded product (e.g., EPO) upon entry of the DNAconstruct into recipient cells.

Transfection of Primary or Secondary Cells and Production of Clonal orHeterogenous Cell Strains

Transfection of cells by the present method is carried out as follows:vertebrate tissue is first obtained; this is carried out using knownprocedures, such as punch biopsy or other surgical methods of obtaininga tissue source of the primary cell type of interest. For example, punchbiopsy is used to obtain skin as a source of fibroblasts orkeratinocytes. A mixture of primary cells is obtained from the tissue,using known methods, such as enzymatic digestion or explantation. Ifenzymatic digestion is used, enzymes such as collagenase, hyaluronidase,dispase, pronase, trypsin, elastase and chymotrypsin can be used.

The resulting primary cell mixture can be transfected directly or it canbe cultured first, removed from the culture plate and resuspended beforetransfection is carried out. Primary cells or secondary cells arecombined with exogenous DNA encoding EPO, to be stably integrated intotheir genomes and, optionally, DNA encoding a selectable marker, andtreated in order to accomplish transfection. The exogenous DNA andselectable marker-encoding DNA can each be present on a separateconstruct (e.g., pXEPO1 and pcDNEO, see FIGS. 1 and 2) or on a singleconstruct (e.g., pE3neoEPO, SEE FIG. 4). An appropriate quantity of DNAto ensure that at least one stably transfected cell containing andappropriately expressing exogenous DNA is produced. In general, 0.1 to500 μg DNA is used.

In one embodiment of the present method of producing transfected primaryor secondary cells, transfection is effected by electroporation, asdescribed in the Examples. Electroporation is carried out at appropriatevoltage and capacitance (and corresponding time constant) to result inentry of the DNA constructs(s) into the primary or secondary cells.Electroporation can be carried out over a wide range of voltages (e.g.,50 to 2000 volts) and corresponding capacitance. As described herein,electroporation is very efficient if carried out at an electroporationvoltage in the range of 250-300 volts and capacitance of 960 μFarads(see Examples 4, 5, 7 and 8). Total DNA of approximately 0.1 to 500 μgis generally used. As described in the Examples, total DNA of 60 μg andvoltage of 250-300 volts with capacitance of 960 μFarads for a timeconstant 14-20 of msec. has been used and shown to be efficient.

In another embodiment of the present method, primary or secondary cellsare transfected using microinjection. See, for instance, Examples 4 and9. Alternatively, known methods such as calcium phosphate precipitation,modified calcium phosphate precipitation and polybrene precipitation,liposome fusion and receptor-mediated gene delivery can be used totransfect cells. A stably, transfected cell is isolated and cultured andsubcultivated, under culturing conditions and for sufficient time, topropagate the stably transfected secondary cells and produce a clonalcell strain of transfected secondary cells. Alternatively, more than onetransfected cell is cultured and subculturated, resulting in productionof a heterogenous cell strain.

Transfected primary or secondary cells undergo a sufficient number ofdoublings to produce either a clonal cell strain or a heterogenous cellstrain of sufficient size to provide EPO to an individual in effectiveamounts. In general, for example, 0.1 cm² of skin is biopsied andassumed to contain 100,000 cells; one cell is used to produce a clonalcell strain and undergoes approximately 27 doublings to produce 100million transfected secondary cells. If a heterogenous cell strain is tobe produced from an original transfected population of approximately100,000 cells, only 10 doublings are needed to produce 100 milliontransfected cells.

The number of required cells in a transfected clonal or heterogenouscell strain is variable and depends on a variety of factors, whichinclude but are not limited to, the use of the transfected cells, thefunctional level of the exogenous DNA in the transfected cells, the siteof implantation of the transfected cells (for example, the number ofcells that can be used is limited by the anatomical site ofimplantation), and the age, surface area, and clinical condition of thepatient. To put these factors in perspective, to deliver therapeuticlevels of EPO in an otherwise healthy 60 kg patient with anemia, thenumber of cells needed is approximately the volume of cells present onthe very tip of the patient's thumb.

Episomal Expression of Exogenous DNA

DNA sequences that are present within the cell yet do not integrate intothe genome are referred to as episomes. Recombinant episomes may beuseful in at least three settings: 1) if a given cell type is incapableof stably integrating the exogenous DNA; 2) if a given cell type isadversely affected by the integration of DNA; and 3) if a given celltype is capable of improved therapeutic function with an episomal ratherthan integrated DNA.

Using the transfection and culturing approaches to gene therapydescribed in Examples 1 and 2, exogenous DNA encoding EPO, in the formof episomes can be introduced into vertebrate primary and secondarycells. Plasmid pE3neoEPO can be converted into such an episome by theaddition DNA sequences for the Epstein-Barr virus origin of replicationand nuclear antigen [Yates, J. L. Nature 319:780-7883 (1985)].Alternatively, vertebrate autonomously replicating sequences can beintroduced into the construct (Weidle, U. H. Gene 73(2):427-437 (1988).These and other episomally derived sequences can also be included in DNAconstructs without selectable markers, such as pXEPO1. The episomalexogenous DNA is then introduced into primary or secondary vertebratecells as described in this application (if a selective marker isincluded in the episome, a selective agent is used to treat thetransfected cells). Similarly, episomal expression of DNA encodinginsulinotropin can be accomplished in vertebrate primary or secondarycells, using the same approach described above with reference to EPO.

Implantation of Clonal Cell Strain or Heterogenous Cell Strain ofTransfected Secondary Cells

The transfected cells produced as described above are introduced into anindividual to whom EPO is to be delivered, using known methods. Theclonal cell strain or heterogenous cell strain is introduced into anindividual, using known methods, using various routes of administrationand at various sites (e.g., renal subcapsular, subcutaneous, centralnervous system (including intrathecal), intravascular, intrahepatic,intrasplanchnic, intraperitoneal (including intraomental), orintramuscular implantation)]. Once implanted in the individual, thetransfected cells produce EPO encoded by the exogenous DNA. For example,an individual who has been diagnosed as anemic is a candidate for a genetherapy cure. The patient has a small skin biopsy performed; this is asimple procedure which can be performed on an out-patient basis. Thepiece of skin, approximately 0.1 cm², is taken, for example, from underthe arm and requires about one minute to remove. The sample isprocessed, resulting in isolation of the patient's cells (in this case,fibroblasts) and genetically engineered to produce EPO. Based on theage, weight, and clinical condition of the patient, the required numberof cells is grown in large-scale culture. The entire process usuallyrequires 4-6 weeks and, at the end of that time, the appropriate numberof genetically-engineered cells is introduced into the individual (e.g.,by injecting them back under the patient's skin). The patient is nowcapable of producing his or her own EPO or additional EPO.

Transfected cells, produced as described above, which containinsulinotropin-encoding DNA are delivered into an individual in whominsulin production, secretion, function and/or sensitivity iscompromised. They are introduced into the individual by known methodsand at various sites of administration (e.g., renal, subcapsular,subcutaneous, central nervous system (including intrathecal),intravascular, intrahepatic, intrasplanchnic, intraperitoneal (includingintraomental) or intramuscular implantation). Once implanted in theindividual, the transfected cells produce insulinotropin encoded by theexogenous DNA. For example, an individual in whom insulin production,secretion or sensitivity is impaired can receive therapy or preventivetreatment through the implantation of transfected cells expressingexogenous DNA encoding insulinotropin produced as described herein. Thecells to be genetically engineered are obtained as described above forEPO, processed in a similar manner to produce sufficient numbers ofcells, and introduced back into the individual.

As this example suggests, the cells used will generally bepatient-specific genetically-engineered cells. It is possible, however,to obtain cells from another individual of the same species or from adifferent species. Use of such cells might require administration of animmunosuppressant, alteration of histocompatibility antigens, or use ofa barrier device to prevent rejection of the implanted cells.

In one embodiment, a barrier device is used to prevent rejection ofimplanted cells obtained from a source other than the recipient (e.g.,from another human or from a non-human mammal such as a cow, dog, pig,goat, sheep or rodent). In this embodiment, transfected cells of thepresent invention are placed within the barrier device, which is made ofa material (e.g., a membrane such as Amicon XM-50) which permits theproduct encoded by the exogenous DNA to pass into the recipient'scirculation or tissues but prevents contact between the cells and therecipient's immune system and thus prevents an immune response to (andpossible rejection of) the cells by the recipient. Alternatively, DNAencoding EPO or insulinotropin can be introduced into an individual bydirect injection, such as into muscle or other appropriate site. In thisembodiment, the DNA construct includes exogenous DNA encoding thetherapeutic product (e.g., EPO, insulinotropin) and sufficientregulatory sequences for expression of the exogenous DNA in recipientcells. After injection into the individual, the DNA construct is takenup by some of the recipient cells. The DNA can be injected alone or in aformulation which includes a physiologically compatible carrier (e.g., aphysiological buffer) and, optionally, other components, such as agentswhich allow more efficient entry of the DNA construct into cells,stabilize the DNA or protect the DNA from degradation.

Uses of Transfected Primary and Secondary Cells and Cell Strains

Transfected primary or secondary cells or cell strains have wideapplicability as a vehicle or delivery system for EPO. The transfectedprimary or secondary cells of the present invention can be used toadminister EPO, which is presently administered by intravenousinjection. When transfected primary or secondary cells are used, there sno need for extensive purification of the polypeptide before it isadministered to an individual, as is generally necessary with anisolated polypeptide. In addition, transfected primary or secondarycells of the present invention produce the therapeutic product as itwould normally be produced.

An advantage to the use of transfected primary or secondary cells of thepresent invention is that by controlling the number of cells introducedinto an individual, one can control the amount of EPO. In addition, insome cases, it is possible to remove the transfected cells if there isnot longer a need for the product. A further advantage of treatment byuse of transfected primary or secondary cells of the present inventionis that production can be regulated, such as through the administrationof zinc, steroids or an agent which affects transcription of theEPO-encoding DNA.

Glucagon-like peptide 1 (GLP-1) and glucagon-like peptide 1 derivatives(GLP-1 derivatives) are additional molecules that can be deliveredtherapeutically using the in vivo protein production and delivery systemdescribed in the present invention. GLP-1 derivatives include truncatedderivatives GLP-1(7-37), GLP-1(7-36), GLP-1(7-35) GLP-1(7-34) and othertruncated carboxy-terminal amidated derivatives and derivatives of GLP-1which have amino acid substitutions, deletions, additions or otheralterations (e.g., addition of a non-amino acid component) which resultin biological activity or stability in the blood which is substantiallythe same as that of a truncated GLP-1 derivative or enhanced biologicalactivity or stability in the blood (greater than that of a truncatedGLP-1 derivative). As used herein, the term GLP-1 derivative includesall of the above-described molecules. The term GLP-1 related peptide, asused herein, includes GLP-1 and GLP-1 derivatives. GLP-1 derivatives,also known as insulinotropins or incretins, are normally secreted intothe circulation by cells in the gastrointestinal tract. In vivo studieshave demonstrated that these peptides function to stimulate insulinsecretion and inhibit glucagon secretion from the endocrine pancreas, aswell as increase insulin sensitivity in peripheral tissues [Goke, R. etal. (1991) Eur. J. Clin. Inv. 21:135-144; Gutniak, M. et al. (1992) NewEngl. J. Med. 326:1316-1322]. Patients with non-insulin dependentdiabetes mellitus (NIDDM) are often treated with high levels of insulinto compensate for their decreased insulin sensitivity. Thus, thestimulation of insulin release and the increase in insulin sensitivityby GLP-1 derivatives would be beneficial for NIDDM patients. Ofparticular importance is the fact that the insulinotropin-inducedstimulation of insulin secretion is strongly dependent on glucoselevels, suggesting that these peptides act in response to increases inblood glucose in vivo to potentiate insulin release and, ultimately,lower blood glucose.

The present invention will now be illustrated by the following examples,which are not intended to be limiting in any way.

EXAMPLES Example 1 Isolation of Fibroblasts

a. Source of Fibroblasts

Human fibroblasts can be obtained from a variety of tissues, includingbiopsy specimens derived from liver, kidney, lung and skin. Theprocedures presented here are optimized for the isolation of skinfibroblasts, which are readily obtained from individuals of any age withminimal discomfort and risk (embryonic and fetal fibroblasts may beisolated using this protocol as well). Minor modifications to theprotocol can be made if the isolation of fibroblasts from other tissuesis desired.

Human skin is obtained following circumcision or punch biopsy. Thespecimen consists of three major components: the epidermal and dermallayers of the skin itself, and a fascial layer that adheres to thedermal layer. Fibroblasts can be isolated from either the dermal orfascial layers.

b. Isolation of Human Fascial Fibroblasts

Approximately 3 cm² tissue is placed into approximately 10 ml of washsolution (Hank's Balanced Salt Solution containing 100 units/mlpenicillin G, 100 μg/ml streptomycin sulfate, and 0.5 μg/ml Fungisone)and subjected to gentle agitation for a total of three 10-minute washesat room temperature. The tissue is then transferred to a 100 mm tissueculture dish containing 10 ml digestion solution (wash solutioncontaining 0.1 units/ml collagenase A, 2.4 units/ml grade II Dispase).

Under a dissecting microscope, the skin is adjusted such that theepidermis is facing down. The fascial tissue is separated from thedermal and epidermal tissue by blunt dissection. The fascial tissue isthen cut into small fragments (less than 1 mm²) and incubated on arotating platform for 30 min at 37° C. The enzyme/cell suspension isremoved and saved, an additional 10 cc of digestion solution is added tothe remaining fragments of tissue, and the tissue is reincubated for 30min at 37° C. The enzyme/cell suspensions are pooled, passed through a15-gauge needle several times, and passed through a Cellector Sieve(Sigma) fitted with a 150-mesh screen. The cell suspension iscentrifuged at 1100 rpm for 15 min at room temperature. The supernatantis aspirated and the disaggregated cells resuspended in 10 ml ofnutrient medium (see below). Fibroblast cultures are initiated on tissueculture treated flasks (Corning) at a density of approximately 40,000cells/cm².

c. Isolation of Human Dermal Fibroblasts

Fascia is removed from skin biopsy or circumcision specimen as describedabove and the skin is cut into small fragments less than 0.5 cm². Thetissue is incubated with 0.25% trypsin for 60 min at 37° C.(alternatively, the tissue can be incubated in trypsin for 18 hrs at 4°C.). Under the dissecting microscope, the dermis and epidermis areseparated. Dermal fibroblasts are then isolated as described above forfascial fibroblasts.

d. Isolation of Rabbit Fibroblasts

The procedure is essentially as described above. Skin should be removedfrom areas that have been shaved and washed with a germicidial solutionand surgically prepared using accepted procedures.

Example 2 Culturing of Fibroblasts

a. Culturing of Human Fibroblasts

When confluent, the primary culture is trypsinized using standardmethods and seeded at approximately 10,000 cells/cm². The cells arecultured at 37° C. in humidified air containing 5% CO₂. Human fibroblastnutrient medium (containing DMEM, high glucose with sodium pyruvate,10-15% calf serum, 20 mM HEPES, 20 mM L-glutamine, 50 units/mlpenicillin G, and 10 μg/ml streptomycin sulfate) is changed twiceweekly.

b. Culturing of rabbit Fibroblasts

The cells are trypsinized and cultured as described for humanfibroblasts. Rabbit fibroblast nutrient medium consists of a 1:1solution of MCDB-110 (Sigma) with 20% calf serum and conditioned medium.Conditioned medium is essentially human fibroblast nutrient medium (with15% calf serum) removed from rabbit fibroblast grown in mass culture for2-3 days.

Example 3 Construction of a Plasmid (pXEPO1) Containing the HumanErythropoietin Gene under the Control of the Mouse MetallothioneinPromoter

The expression plasmid pXEPO1 has the hEPO gene under thetranscriptional control of the mouse metallothionein (mMT) promoter.pXEPO1 is constructed as follows: Plasmid pUC19 (ATCC #37254) isdigested with KpnI and BamHI and ligated to a 0.7 kb KpnI-BgIII fragmentcontaining the mouse metallothionein promoter [Hamer, D. H. and Walling,M., J. Mol. Appl. Gen., 1:273-288 (1982). This fragment can also beisolated by known methods from mouse genomic DNA using PCR primersdesigned from analysis of mMT sequences available from Genbank; i.e.MUSMTI, MUSMTIP, MUSMTIPRM]. The resulting clone is designated pXQM2.

The hEPO gene was isolated by from a bacteriophage lambda clonecontaining the entire hEPO gene. This bacteriophage was isolated byscreening a human Sau3A-partial genomic DNA library (Stratagene)constructed in the lambda vector LAMBDA DASH with 0.77 kb fragment ofthe human gene. This 0.77 kb fragment was amplified from human genomicDNA using the primers shown below in the polymerase chain reaction(PCR).

Human EPO PCR Primers

Oligo hEPO-1: 5'GTTTGCTCAGCTTGGTGCTTG (Seq. ID No. 1) (positions2214-2234 in the Genbank HUMERPA sequence)

Oligo hEPO-2: 5'TCAAGTTGGCCCTGTGACAT (Seq. ID No. 2) (positions2986-2967 in the Genbank HUMERPA sequence)

The amplified fragment, encompassing exons 4 and 5 of the human EPOgene, was radiolabelled and used to screen the human genomic DNAlibrary. Phage with a 5.4 kb HindIII-BamHI fragment containing theentire human EPO gene were assumed to contain the entire gene, based onpublished DNA sequence and restriction enzyme mapping data [Lin, F.-K.,et al., Proc. Natl. Acad. Sci. USA, 82:7580-7584 (1985)].

A 4.8 kb BstEII-BamHI fragment (BstEII site is at position 580 inGenbank HUMERPA sequence; the BamHI site is 4.8 kb 3' of this site,outside of the sequenced region) was isolated from the bacteriophageclone. The purified fragment is made blunt-ended by treatment with theKlenow fragment of E. coli DNA polymerase and ligated to HincII digestedpXQM2, which cuts in the pUC19-derived polylinker adjacent to the 3'side of the subcloned mMT promoter. One orientation, in which theablated BstEII site is proximal to the mMT promoter, was identified byrestriction mapping and designated pXEPO1 (FIG. 1).

Example 4 Transfection of Primary and Secondary Fibroblasts withExogenous DNA and a Selectable Marker Gene by Electroporation andMicroinjection

To prepare cells for electroporation, exponentially growing or earlystationary phase fibroblast are trypsinized and rinsed from the plasticsurface with nutrient medium. An aliquot of the cell suspension isremoved for counting, and the remaining cells are subjected tocentrifugation as described above. The supernatant is aspirated and thepellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cellsare recentrifuged, the supernatant aspirated, and the cells resuspendedin electroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Supercoiled plasmid DNA is added to a sterile cuvette with a 0.4 cmelectrode gap (Bio-Rad). The final DNA concentration is generally atleast 120 μg/ml. 0.5 ml of the cell suspension (containing approximately1.5×10⁶ cells) is then added to the cuvette, and the cell suspension andDNA solutions are gently mixed. Electroporation is performed with aGene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960μF and 250-300 V, respectively. As voltage increases, cell survivaldecreases, but the percentage of surviving cells that stably incorporatethe introduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (as above with 15% calf serum) in a 10cm dish and incubated as described above. The following day, the mediais aspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hrs. Subculture of cells to determine cloning efficiencyand to select for G418-resistant colonies is performed the followingday. Cells are trypsinized, counted and plated; typically, fibroblastsare plated at 10³ cells/10 cm dish for the determination of cloningefficiency and at 1-2×10⁴ cells/10 cm dish for G418 selection.

Human fibroblasts are selected for G418 resistance in medium consistingof 300-400 μg/ml G418 (Geneticin, disulfate salt with a potency ofapproximately 50%; Gibco) in fibroblasts nutrient media (with 15% calfserum). Cloning efficiency is determined in the absence of G418. Theplated cells are incubated for 12-14 days, at which time colonies arefixed with formalin, stained with crystal violet and counted (forcloning efficiency plates) or isolated using cloning cylinders (for G418plates). Electroporation and selection of rabbit fibroblasts isperformed essentially as described for human fibroblasts, with theexception of the nutrient media used. Rabbit fibroblasts are selectedfor G418 resistance in medium containing 1 mg/ml G418.

Fibroblasts were isolated from freshly excised human foreskins. Cultureswere seeded at 50,000 cells/cm² in DMEM+10% calf serum. When culturesbecame confluent fibroblasts were harvested by trypsinization andtransfected by electroporation. Electroporation conditions wereevaluated by transfection with the plasmid pcDNEO. A representativeelectroporation experiment using near optimal conditions (60 μg ofplasmid pcDNEO at an electroporation voltage of 250 volts and acapacitance setting of 960 μFarads) resulted in one G418^(r) coloney per588 treated cells (0.17% of all cells treated), or one G418^(r) colonyper 71 clonable cells (1.4%).

When nine separate electroporation experiments at near optimalconditions (60 μg of plasmid pcDNEO at an electroporation voltage of 300volts and a capacitance setting of 960 μFarads) were performed, anaverage of one G418^(r) colony per 1,899 treated cells (0.05%) wasobserved, with a range of 1/882 to 1/7,500 treated cells. Thiscorresponds to an average of one G418^(r) colony per 38 clonable cells(2.6%).

Low passage primary human fibroblasts were converted to hGH expressingcells by co-transfection with plasmids pcDNEO and pXGH5 [Selden, R. F.et al., Mol. Cell. Biol., 6:3173-3179 (1986)]. Typically, 60 μg of anequimolar mixture of the two plasmids were transfected at near optimalconditions (electroporation voltage of 300 volts and a capacitancesetting of 960 μFarads). The results of such an experiment resulted inone G418^(r) colony per 14,705 treated cells.

hGH expression data for these and other cells isolated under identicaltransfection conditions are summarized below. Ultimately, 98% of allG418^(r) colonies could be expanded to generate mass cultures.

    ______________________________________                                        Number of G418.sup.r Clones                                                                   154                                                             Analyzed                                                                      Number of G418.sup.r /hGH  65                                                 Expressing Clones                                                             Average hGH Expression  2.3 μg hGH/10.sup.6 Cells/24 hr                    Level                                                                         Maximum hGH Expression 23.0 μg hGH/10.sup.6 Cells/24 hr                    Level                                                                       ______________________________________                                    

Stable transfectants also have been generated by electroporation ofprimary or secondary human fibroblasts with pXGH301, a DNA construct inwhich the neo and hGH genes are present on the same plasmid molecule(Example 3). For example, 1.5×10⁶ cells were electroporated with 60 μgpXGH301 at 300 volts and 960 μFarads. G418 resistant colonies wereisolated from transfected secondary fibroblasts at a frequency of 652G418 resistant colonies per 1.5×10⁶ treated cells (1 per 2299 treatedcells). Approximately 59% of these colonies express hGH.

Primary and secondary human fibroblasts can also be transfected bydirect injection of DNA into cell nuclei. The ability of primary andsecondary human foreskin fibroblasts to be stably transfected by thismethod has not been previously reported. The 8 kb HindIII fragment fromplasmid RV6.9h (Zheng, H. et al., Proc. Natl. Acad. Sci. USA 88:188067-8071 (1991)) was purified by gel electrophoresis and passagethrough an anion exchange column (QIAGEN Inc.). DNA at (10 μg/ml) wasinjected into primary or secondary human foreskin fibroblasts using 0.1μm outer diameter glass needles. 41 G418^(r) clones were isolated afterinjection of 2,000 cells (1 in 49 starting cells).

hGH expressing clones were also generated by microinjection. PlasmidpXGH301 (FIG. 3) was linearized by ScaI digestion (which cuts oncewithin the amp^(r) gene in the pUC12 backbone), purified by passagethrough an anion exchange column (QIAGEN Inc.), and injected intosecondary human foreskin fibroblasts using 0.1 μm outer diameter glassneedles. Several DNA concentrations were used, ranging from 2.5-20 μgpXGH301/ml. Twenty G418 resistant clones were isolated aftermicroinjection into 2,100 cells (1 in 105 starting cells). The fractionof G418^(r) cells, is approximately 1% of all cells treated. Nine of 10clones analyzed were expressing hGH, with average hGH expression being0.6 μg/10⁶ cells/24 hr for clones isolated in this experiment, and 3clones were expanded for studying long-term expression of hGH. All 3were expressing hGH stably, with hGH still being produced through 33,44, and 73 mpd for the 3 strains, respectively.

Example 5 In vitro hEPO Production by Transfected Secondary Human andRabbit Skin Fibroblasts

1. Human Skin Fibroblasts

Fibroblasts were isolated from freshly excised human skin fibroblastsand cultured in DMEM+15% calf serum. Electroporation (250 volts, 960μFarads) with 60 μg of an equimolar mixture of pcDNEO and pXEPO1 wasperformed on passage 1 cells and treated cells were selected inG418-containing medium (300 μg/ml G418). Colonies were isolated andexpanded using standard methods. Data derived from an analysis offifty-six individual clones is shown in Table 1 below. Cells weremaintained in G418 (300 μg/ml G418) in DMEM+15% calf serum andsubcultured at a seeding density of 10,000 cells/cm². Culture medium waschanged 24 hr prior to harvesting the cells for passaging. At the timeof passage, an aliquot of the culture medium was removed for hEPO assayand the cells were then harvested, counted, and reseeded. hEPOconcentration in the medium was determined using a commerciallyavailable ELISA (R & D Systems). hEPO levels are expressed as mU/10⁶cells/24 hr., and expression levels ranged from 69 to 55,591 mU/10⁶cells/24 hr. 19% of all G418-resistant colonies expressed detectablelevels of hEPO.

                  TABLE 1                                                         ______________________________________                                        hEPO EXPRESSION IN FIFTY-SIX INDEPENDENT                                        SECONDARY HUMAN FIBROBLAST CLONES ISOLATED                                    BY CO-TRANSFECTION WITH pcDNEO AND pXEPO1                                         HEPO Expression Level                                                     (mU/10.sup.6 cells/24 hr) Number of Clones                                  ______________________________________                                         <1,000          10                                                              1,000-10,000 28                                                              10,000-50,000 17                                                              >50,000  1                                                                  ______________________________________                                    

Clonally derived human fibroblasts isolated by co-transfection withpcDneo and pXEPO1 were analyzed for the glycosylation state of secretedhEPO. Media was collected from hEPO producing cells andimmunoprecipitated with a mouse monoclonal antibody (GenzymeCorporation) specific for human erythropoietin. The immunoprecipitatedmaterial was subject to electrophoresis on a 12.5% polyacrylamide geland transferred to a PVDF membrane (Millipore). The membrane was probedwith the same anti-hEPO monoclonal antibody used for immunoprecipitationand was subsequently treated with an HRP-conjugated sheep anti-mouse IgGantisera (Cappel), followed by luminescent detection (ECL Westernblotting detection kit; Amersham) to visualize hEPO through theproduction of a fluorescent product.

As shown in FIG. 5A, a molecule with a molecular ass of approximately 34kd reacts with an antibody specific for human erythropoietin. This isthe expected size for naturally occurring, fully glycosylated humanerythropoietin.

hEPO produced by transfected human fibroblast clones was furtheranalyzed to determine if the secreted material had both N- and O-linkedglycosylation characteristic of natural human erythropoietin isolatedfrom urine or recombinant hEPO produced by chinese hamster ovary cells.FIG. 5B shows a Western blot of the untreated cell supernatant (lane 1),the supernatant treated with endoglycosidase-F [(New England Nuclear);lane 2], the supernatant treated with neuraminidase [Genzyme); (lane3)], and the supernatant treated with O-glycanase [(Genzyme); (lane 4)].Treatment with endoglycosidase-F results in a shift in molecular weightfrom 34 kd to approximately 27 kd. Treatment with neuraminidase resultsin a barely detectable shift in band position, while treatment withO-glycanase further shifts the size of the immunoreactive band down toapproximately 18.5 kd. These results are indistinguishable from thoseobtained with natural human erythropoietin isolated from urine orrecombinant hEPO produced by Chinese hamster ovary cells (Browne, J. K.et al., Cold Spring Harbor Symp. Quant. Biol. 51:693-702 (1986)).

2. Rabbit Fibroblasts

Fibroblasts were isolated from freshly excised rabbit skin and culturedin DMEM 10% calf serum. Electroporation (250 volts, 960 μFarads) with 60μg of an equimolar mixture of pcDNEO and pXEPO1 was performed andtreated cells were selected in G418-containing rabbit fibroblast growthmedium (1 mg/ml G418; Example 2). Colonies were isolated and expandedusing standard methods, and the resulting secondary cell strains wereanalyzed for hEPO expression. Data derived from forty-nine independentrabbit fibroblast clones is shown in Table 2 below. Expression levels inthese clones ranged from 43 to 2,900,000 mU/10⁶ cells/24 hr., and 72% ofall G418-resistant clones expressed detectable levels of hEPO.

                  TABLE 2                                                         ______________________________________                                        hEPO EXPRESSION IN FORTY-NINE INDEPENDENT                                       SECONDARY RABBIT FIBROBLAST CLONES ISOLATED                                   BY CO-TRANSFECTION WITH pcDNEO AND pEEPO                                          hEPO Expression Level                                                     (mU/10.sup.6 cells/24 hr) Number of Clones                                  ______________________________________                                         <1,000          1                                                               1,000-10,000 3                                                               10,000-50,000 7                                                                50,000-500,000 19                                                            >500,000 19                                                                 ______________________________________                                    

Example 6 Construction of a Plasmid Containing Both the Human EPO Geneand the Neomycin Resistance Gene

A 6.9 kb HindIII fragment extending from positions 11,960-18,869 in theHPRT sequence [Genbank entry HUMHPRTB; Edwards, A. et al., Genomics,6:593-608 (1990)] and including exons 2 and 3 of the HPRT gene, issubcloned into the HindIII site of pUC12. The resulting clone is cleavedat the unique Xhol site in exon 3 of the HPRT gene fragment and the 1.1kb SaII-XhoI fragment containing the neo gene from pMC1NEO (Stratagene)is inserted, disrupting the coding sequence of exon 3. One orientation,with the direction of neo transcription opposite that of HPRTtranscription was chosen and designated pE3Neo. pE3neo has a unique XhoIsite at the junction of HPRT sequences and the 5' side of the neo gene.pE3neo is cut with XhoI and made blunt-ended by treatment with theKlenow fragment of E. coli DNA polymerase.

To insert the hEPO gene into the neo selection plasmid pE3Neo, a 5.1 kbEcoRI-HindIII fragment was isolated from plasmid pXEPO1 (Example 3; FIG.1). The EcoRI site is located adjacent to the 5' side of the mMTpromoter, and the HindIII site is located 5.1 kb away, 3' to the hEPOcoding region. The purified Fragment is made blunt-ended by treatmentwith Klenow fragment of E. coli DNA polymerase and ligated to the XhoIdigested and blunt-ended pE3neo fragment described above. Aftertransformation into E. coli, a plasmid with one copy of the mMT-hEPOfragment inserted into pE3neo was identified by restriction enzymeanalysis in which the hEPO gene is transcribed in the same orientationas the adjacent neo gene. This plasmid was designated pE3neoEPO. Inaddition to allowing direct selection of hEPO expressing G418^(r)clones, this fragment may also be used in gene targeting to direct theintegration of the hEPO gene to the human HPRT locus.

Example 7 Isolation of Human Fibroblast Clones Expressing hEPO Gene anda Selectable Marker (pE3neoEPO)

Fibroblasts were isolated from freshly excised human skin fibroblastsand cultured in DMEM+15% calf serum. Electroporation (250 volts, 960μFarads) with 60 μg of supercoiled pE3neoEPO was performed on passage 1cells and treated cells were selected in G418-containing medium (300μg/ml G418). Colonies were isolated and expanded using standard methods.Data derived from an analysis of twenty-six individual clones is shownin Table 3 below. Cells were maintained in G418 (300 μg/ml G418) inDMEM+15% calf serum and subcultured at a seeding density of 10,000cells/cm². Culture medium was changed 24 hr prior to harvesting thecells for passaging. At the time of passage an aliquot of the culturemedium was removed for hEPO assay and the cells were then harvested,counted, and reseeded. hEPO concentration in the medium was determinedusing a commercially available ELISA (R and D Systems). hEPO levels areexpressed as mU hEPO/10⁶ cells/24 hr, and expression levels ranged from240 to 961,620 mU/10⁶ cells/24 hr. 89% of all G418-resistant clonesexpressed detectable levels of hEPO.

                  TABLE 3                                                         ______________________________________                                        hEPO EXPRESSION IN TWENTY-SIX INDEPENDENT                                       SECONDARY HUMAN FIBROBLAST CLONES ISOLATED                                    BY TRANSFECTION WITH pE3neo-EPO                                                   hEPO Expression Level                                                     (mU/10.sup.6 cells/24 hr) Number of Clones                                  ______________________________________                                         <1,000          2                                                               1,000-10,000 2                                                               10,000-50,000 9                                                                50,000-500,000 12                                                            >500,000 1                                                                  ______________________________________                                    

hEPO expressing human fibroblast clones are also isolated byelectroporation with 60 g of HindIII digested pE3neoEPO. hEPO expressingrabbit fibroblast clones are isolated using plasmid pE3neoEPO underidentical transfection conditions, with the exception that rabbitfibroblast clones are selected in rabbit fibroblast growth medium(Example 2) containing 1 mg/ml G418.

Example 8 Isolation of Transfectants in the Absence of Selection

The high frequency of transfection in human fibroblasts (greater than 1%stable transfectant per clonable cell; Example 4) indicates that itshould be possible to isolate cell clones that have stably incorporatedexogenous DNA without the use of a selective agent. Stable transfectionof primary fibroblasts with the plasmid pXEPO1 should render recipientfibroblasts capable of secreting human erythropoietin into thesurrounding medium. Therefore, an ELISA for hEPO (or for any expressedprotein of therapeutic interest) can be used as a simple and rapidscreen for transfectants. Alternatively, it should be possible todetermine the true frequency of stable integration of exogenous DNAusing a screening method such as PCR which does not necessarily rely onexpression of transfected DNA.

1. Primary Human Fibroblasts

Approximately 2.0×10⁶ human cells that were freshly dissociated fromtissue are electroporated with 60 μg of pXEPO1 at 300 volts, 960μFarads. Cells are plated immediately in a 100 mm tissue culture dishcontaining 10 ml of prewarmed medium and incubated at 37° C. in ahumidified 5% CO₂ atmosphere. Two days following transfection, 5×10³cells are subcultured into a 24 well cloning plate (Bellco Glass Co.).Each well of the 24 well plate contained 16 smaller wells (384wells/plate). Eight days after plating into the 24 large wells, media isscreened for hEPO expression via ELISA. A second, confirming assay, isdone in which media from wells exhibiting hEPO expression is aspiratedout, replaced with fresh media and assayed for hEPO 24 hours later.Colony counts at this stage should reveal approximately 10 colonies perlarge well.

Individual colonies in each of the 16 small wells within one of thehEPO-positive larger wells are trypsinized and transferred to wells of a96 well plate. Three days later each of those wells are assayed for hEPOexpression. Cells from hEPO positive cells are expanded for furtherstudy. This experiment may also be performed using secondary humanforeskin fibroblasts.

2. Primary Rabbit Fibroblasts

Passage 1 rabbit skin cells were transfected with pXEPO1. Theelectroporation conditions were identical to the human tissueelectroporation described above. 1×10³ cells are subcultured into a 384well plate. Seven days later hGH assays are performed on media from eachof the 24 large wells. Cells in each of the small wells in hEPO-positivelarge wells are trypsinized and transferred to wells of a 96 well plate.Three days later each of these wells are assayed for hEPO expression.Cells from hEPO positive cells are expanded for further study. Thisexperiment may also be performed using secondary rabbit skinfibroblasts.

Example 9 Stable Transfection of Primary Human Fibroblasts byMicroinjection

Direct injection of DNA into cell nuclei is another method for stablytransfecting cells. The ability of primary and secondary human foreskinfibroblasts to be stably transfected by this method is described inExample 4, but has not been previously reported in the literature. The13.1 kb HindIII fragment from plasmid pE3neoEPO is purified by gelelectrophoresis and passed through an anion exchange column (QIAGENInc.). This fragment contains the human EPO and bacterial neo genes,flanked on both sides with human HPRT sequences. DNA at (10 μg/ml) isinjected into primary or secondary human foreskin fibroblasts using 0.1μm diameter glass needles. G418^(r) clones are isolated approximately12-14 days after injection. Alternatively, the total HindIII digest ofpE3neoEPO, as well as linearized or supercoiled pE3neoEPO may beinjected to isolate hEPO expressing cells.

Example 10 Expression of Biologically Active Human Erythropoietin inMice

The mouse provides a valuable system to study implants of geneticallyengineered cells for their ability to deliver therapeutically usefulproteins to an animal's general circulation. The relativeimmuneincompetence of nude mice allow xenogeneic implants to retainbiologic function and may allow certain primary and secondary rabbitfibroblasts to survive in vivo for extended periods.

For implantation of cells into the subrenal capsule, mice are givenintraperitoneal injection of Avertin at a dose of 0.0175 ml/g bodyweight. The kidney (generally the left kidney) is approached through an8-10 mm incision made approximately 3 mm below the rib cage. The skin,abdominal musculature, peritoneum, and peri-renal fascia are retractedto expose the kidney. A small forcep is used to pull the kidney out ofthe abdominal cavity. A 27-gauge hypodermic needle is used to make asmall opening in the renal capsule. Using a 20-gauge I.V. catheter,cells to be implanted (typically 3 million cells in a volume of 5-10 μl)are withdrawn into a 1 ml syringe and slowly ejected under the renalcapsule. Care is taken to ensure that the cells are released distal tothe opening in the renal capsule. The incision is closed with one staplethrough the musculature and the skin. Blood is collected after placingthe mouse in a large beaker containing methoxyflurane until lightanesthesia is achieved. The tip of a Pasteur pipette is placed betweenthe eye and the periorbital space to collect blood from the orbitalsinus. Serum hEPO levels are determined using a commercially availablekit (R and D Systems). An aliquot of blood is also drawn into EDTAcoated capillary tubes (Statspin, Norwood, Mass.) for determination ofhematocrit levels.

A clonal strain of rabbit skin fibroblasts was isolated by the methodsdescribed in Example 5. One clone, designated RF115-D4, was determinedto be stably transfected with the human EPO gene and expressedapproximately 786,000 mU hEPO/10⁶ cells/24 hr. Three million cells wereimplanted into the subrenal capsule in each of six nude mice.Approximately 400 μl of blood was drawn on days 1 and 7 afterimplantation and on every other day thereafter until day 21. During thistime an equal volume of saline solution was injected after bleeding toprevent hypotonic shock. Blood was drawn weekly thereafter until day 63.An identical bleeding schedule was used on ten mice that had no cellsimplanted. FIG. 6A shows the effect of these treatments on bloodhematocrit (HCT), a commonly used indicator of red blood cell number, inimplanted and control animals. In control animals, HCT dropsdramatically by day 7, followed by a return to approximately normallevels by day 15. In contrast, animals receiving implants of the hEPOexpressing cells showed elevated HCT levels by day 7. HCT remainedelevated through day 63, reaching a peak of 64%, or 1.4 times higherthan the day 1 level of 45%, on day 35 after implantation. As shown inFIG. 6B, immunoreactive hEPO was readily detectable in the blood ofimplanted animals (the sensitivity of the hEPO ELISA has been determinedto be 2 mU/ml by the kit's manufacturer (R and D Systems) and endogenousmouse EPO shows no cross-reactivity with the antibodies used in theELISA kit). hEPO levels in the implanted animals dropped gradually, from29 to 9 mU/ml, from days 7 to 63 post-implantation.

This Example clearly demonstrates that normal skin fibroblasts that havebeen genetically engineered to express and secrete hEPO can: 1) survivein vivo to deliver hEPO to an animals systemic circulation for up to 2months, and 2) the hEPO produced is biologically functional, serving toprevent the drop in hematocrit observed in the frequently bled controlanimals, and resulting in a net increase in HCT even when animals werechallenged with a bleeding schedule that produces an anemic response incontrol animals.

Example 11 Expression of GLP-1(7-37) from Secondary Human SkinFibroblasts Strains after Transfection with a GLP-1(7-37) ExpressionPlasmid

The portion of GLP-1 from amino acid residues 7 to 37 [GLP-1(7-37);encoded by base pairs 7214 to 7306 in Genbank sequence HUMGLUCG2] hasbeen demonstrated to have insulinotropin activity in vivo. PlasmidpXGLP1 is constructed such that the GLP-1(7-37) moiety is fused at itsN-terminus to a 26-amino acid signal peptide derived from human growthhormone for efficient transport through the endoplasmic reticulum. Thefusion protein is cleaved immediately C-terminal to residue 26 prior tosecretion, such that the secreted product consists solely of residues7-37 of GLP-1. Expression of the signal peptide: GLP-1(1-37) fusionprotein is controlled by the mouse metallothionein promoter.

Plasmid pXGLP1 is constructed as follows: Plasmid PXGH5 [Selden, R. F.et al., Mol. Cell. Biol. 6:3173-3179 (1986)] is digested with SmaI andligated to a double-stranded oligonucleotide containing a BgIII site(BgIII linkers; New England Biolabs). The ligated product is digestedwith BgIII and EcoRI and the 0.32 kb fragment corresponding to the3'-untranslated region of the human growth hormone gene is isolated(with a BgIII linker attached to the SmaI site lying at position 6698 inGenbank entry HUMGHCSA). The hGH fragment can also be isolated by knownmethods from human genomic DNA using PCR primers designed to amplify thesequence between positions 6698 to 7321 in Genbank entry HUMGHCSA. A1.45 EcoRI-BgIII fragment containing the mouse metallothionein (mMT)promoter [Hamer, D. H. and Walling, M., J. Mol. Appl. Gen., 1:273-288(1982)] is next isolated. The mouse metallothionein promoter may beisolated by known methods from mouse genomic DNA using PCR primersdesigned from analysis of mMT sequences available from Genbank (i.e.Genbank entries MUSMTI, MUSMTIP, and MUSMTIPRM). Plasmid pUC19 (ATCC#37254) is digested with EcoRI and treated with bacterial alkalinephosphatase. The treated plasmid is ligated with the hGH and mMTfragments described above. The resulting plasmid has a single copy ofeach the mouse metallothionein promoter and the 3'untranslated region ofhGH joined at a BgIII site. This plasmid, designated pX1 is digestedwith BgIII and the full-length linear product is purified by gelelectrophoresis.

Oligonucleotides 11.1 and 11.2 are used to amplify a DNA sequenceencoding amino acids 7-37 of GLP-1 from human genomic DNA by PCR. Theamplified product (104 bp) is purified and mixed with pXGH5 andoligonucleotides 11.2, 11.3, 11.4, and 11.5 and subject to PCR.Oligonucleotides 11.3 and 11.4 are complementary and correspond to thedesired junction between the hGH signal peptide and GLP-1 amino acidresidue 7. The 500 base pair amplification product contains5'-untranslated, exon 1, intron 1, and part of exon 2 sequences from hGH(nucleotides 5168 to 5562 in Genbank entry HUMGHCSA) fused to a sequenceencoding GLP-1 residues 7-37 followed by a stop codon. The fragment, bydesign, is flanked on both ends by BamHI sites.

The fragment is cleaved with BamHI and ligated to the BgIII digest ofpX1 described above. Ligation products are analysed to identify thosewith one copy of the hGH-GLP-1(7-37) fusion product inserted at theBgIII site separating the mMT promoter and the 3'-untranslated hGHsequence in pX1, such that GLP-1 residue 37 is distal to the mMTpromoter.

Oligonucleotides for Amplification of hGH-GLP-1(7-37) Fusion Gene

    11.1                                                                             5'CATGCTGAAG GGACCTTTAC CAGT   (Seq. ID No. 3)                                - 11.2 5'TTGGATCCTT ATCCTCGGCC TTTCACCAGC CA (Seq. ID No. 4)                       BamHI                                                                    - 11.3 5'GGCTTCAAGA GGGCAGTGCC CATGCTGAAG GGACCTTTAC CAGT (Seq. ID No.                                                 5)                                   - 11.4 5'ACTGGTAAAG GTCCCTTCAG CATGGGCACT GCCCTCTTGA AGCC (Seq. ID No.                                                 6)                                   - 11.5 5'AAGGATCCCA AGGCCCAACT CCCCGAAC (Seq. ID No. 7)                            BamHI                                                                    - 11.6 5'TTGGATCCTT ATCGGCC TTTCACCAGC CA (Seq. ID No. 8)                          BamHI                                                             

Alternatively, the small sizes of the signal peptide and GLP-1 moietiesneeded allow complete fusion genes to be prepared synthetically. DNAencoding the signal peptides of the LDL receptor (amino acid residues1-21), preproglucagon (amino acid residues 1-20), or human growthhormone (amino acid residues 1-26) may be synthesized by known methodsand ligated in vitro to similarly synthesized DNA encoding amino acids7-37 or 7-36 of GLP-1 (followed immediately by a stop codon). Thesequences necessary to design and synthesize these molecules are readilyavailable in Genbank entries HUMLDLR01 (human LDL receptor), HUMGLUCG2(human GLP-1 and glucagon sequences), and HUMGHCSA (human growthhormone). The ligated product may be inserted into a suitable mammalianexpression vector for use in human fibroblasts. Plasmid pMSG (PharmaciaLKB Biotechnology, Piscataway, N.J.) is suitable for this purpose,having 5' and 3'untranslated sequences, a splice site, a polyA additionsite, and an enhancer and promoter for use in human skin fibroblasts.Alternatively, the ligated product may be synthesized with anappropriate 5'-untranslated sequence and inserted into plasmid pX1described above.

A second insulinotropic GLP-1 derivative, GLP-1(7-36), can be expressedby substituting oligonucleotide 11.6 for oligonucleotide 11.2 describedabove. All subsequent cloning operations described above forconstruction of pXGLP1 are followed, such that the final product islacking the C-terminal glycine residue characteristic of GLP-1(7-37).Alternatively, this terminal glycine residue may be removed in vivo bythe activity of a peptidyl-glycine alpha-amidating enzyme to produce theinsulinotropin GLP-1(7-36) amide.

Plasmid pXGLP1 is co-transfected into primary human skin fibroblastswith plasmid pcDNEO exactly as described for pXEPO1 and pcDNEO inExample 5. Clones are selected in G418 containing medium, transferred to96-well plates, and assayed for GLP-1(7-37) activity or immunoreactivityin cell supernatants. GLP-1(7-37) activity is determined by incubationof cell supernatants with rat insulinoma RINm5F cells and measuring theability of the supernatants to induce insulin secretion from these cellsusing a commercially available insulin radioimmunoassay (Coat-a-CountInsulin, DPC, Los Angeles, Calif.). GLP-1(7-37) antigen is determinedusing a commercially available antisera against GLP-1 (PeninsulaLaboratories, Belmont, Calif.). GLP-1(7-37) positive clones are expandedfor implantation into nude mice as described in Example 10 and bloodsamples are taken to monitor serum human GLP-1(7-37) levels.

In vivo activity is monitored in fasting animals by determining theinsulinogenic index after intraperitoneal injection of glucose (1 mgglucose per gram of body weight). Typically, implanted and non-implantedgroups of 32 mice are fasted overnight, and 28 are injected withglucose. After injection, the 28 mice are arbitrarily assigned to sevengroups of four, and blood sampling (for serum glucose and insulin) isperformed on each group at 5, 10, 20, 30, 45, 60, or 90 minutespost-injection, with the non-glucose injected group serving as a fastingcontrol. Increases in the postinjection insulinogenic index (the rationof insulin to glucose in the blood) in animals receiving GLP-1(7-37)expressing cells over non-implanted animals provides in vivo support forthe insulinotropic activity of the expressed peptide.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingnot more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Statement Regarding Correspondence of Sequence Information (Paper Copyand Disk)

A sequence listing in computer readable form and in paper form are beingfiled with this application. As required by 37 C.F.R. 1.821 (f),Applicants' Attorney hereby states that the content of the SequenceListing in paper from and of the computer readable form of the "SequenceListing" are the same.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 8                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GTTTGCTCAG CTTGGTGCTT G           - #                  - #                      - #21                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TCAAGTTGGC CCTGTGACAT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - CATGCTGAAG GGACCTTTAC CAGT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TTGGATCCTT ATCCTCGGCC TTTCACCAGC CA       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GGCTTCAAGA GGGCAGTGCC CATGCTGAAG GGACCTTTAC CAGT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - ACTGGTAAAG GTCCCTTCAG CATGGGCACT GCCCTCTTGA AGCC   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AAGGATCCCA AGGCCCAACT CCCCGAAC         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TTGGATCCTT ATCGGCCTTT CACCAGCCA         - #                  - #                29                                                                    __________________________________________________________________________

We claim:
 1. A method of expressing erythropoietin in a mammal,comprising the steps of:a) obtaining a source of primary cells from amammal; b) transfecting primary cells obtained in (a) with a DNAconstruct comprising exogenous DNA encoding erythropoietin andadditional DNA sequences sufficient for expression of the exogenous DNAin the primary cells, thereby producing transfected primary cells whichexpress the exogenous DNA encoding erythropoietin; c) culturing atransfected primary cell produced in (b), which expresses the exogenousDNA encoding erythropoietin, under conditions appropriate forpropagating the transfected primary cell which expresses the exogenousDNA encoding erythropoietin, thereby producing a clonal cell strain oftransfected secondary cells from the transfected primary cell; d)culturing the clonal cell strain of transfected secondary cells producedin (c) under conditions appropriate for and sufficient time for theclonal cell strain of transfected secondary cells to undergo asufficient number of doublings to provide a sufficient number oftransfected secondary cells to produce erythropoietin; and e)introducing transfected secondary cells produced in (d) into a mammal ofthe same species as the mammal from which the primary cells wereobtained in sufficient number to express erythropoietin in the mammal.2. The method of claim 1 wherein the primary cells are selected from thegroup consisting of: fibroblasts, keratinocytes, epithelial cells,endothelial cells, glial cells, neural cells, lymphocytes, bone marrowcells, hepatocytes, and precursors thereof.
 3. A method of providingerythropoietin to a mammal, comprising the steps of:a) obtaining asource of primary cells from a mammal; b) producing a population ofsecondary cells from the primary cells obtained in (a); c) transfectingsecondary cells produced in (b) with a DNA construct comprisingexogenous DNA encoding erythropoietin and additional DNA sequencessufficient for expression of the exogenous DNA in the secondary cells,thereby producing transfected secondary cells which express theexogenous DNA encoding erythropoietin; d) culturing a transfectedsecondary cell produced in (c), which expresses the exogenous DNAencoding erythropoietin, under conditions appropriate for propagatingthe transfected secondary cell which expresses the exogenous DNAencoding erythropoietin, thereby producing a clonal cell strain oftransfected secondary cells from the transfected secondary cell; e)culturing the clonal cell strain of transfected secondary cells producedin (d) under conditions appropriate for and sufficient time for theclonal cell strain of transfected secondary cells to undergo asufficient number of doublings to provide a sufficient number oftransfected secondary cells to produce erythropoietin; and f)introducing transfected secondary cells produced in (e) into a mammal ofthe same species as the mammal from which the primary cells wereobtained in sufficient number to provide erythropoietin to the mammal.4. The method of claim 3 wherein the primary cells are selected from thegroup consisting of: fibroblasts, keratinocytes, epithelial cells,endothelial cells, glial cells, neural cells, lymphocytes, bone marrowcells, hepatocytes and precursors thereof.
 5. A method of producingerythropoietin in a mammal, comprising the steps of:a) obtaining asource of primary cells from a mammal; b) transfecting primary cellsobtained in (a) with exogenous DNA encoding erythropoietin andoperatively linked to DNA sequences of non-retroviral origin sufficientfor expression of the exogenous DNA in transfected-primary cells,thereby producing a mixture of primary cells which includes transfectedprimary cells which express the exogenous DNA encoding erythropoietin;c) culturing the product of (b) under conditions appropriate forpropagation of transfected primary cells which express the exogenous DNAencoding erythropoietin, thereby producing a heterogenous cell strain oftransfected secondary cells of mammalian origin which express theexogenous DNA encoding erythropoietin; and d) introducing transfectedsecondary cells produced in (c) into a mammal of the same species as themammal from which the primary cells were obtained in sufficient numberto produce erythropoietin in the mammal.
 6. The method of claim 5,wherein the primary cells are selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, lymphocytes, bone marrow cells, hepatocytes, andprecursors thereof.
 7. A method of providing erythropoietin to a mammal,comprising the steps of:a) obtaining a source of primary cells from amammal; b) producing a population of secondary cells from the primarycells obtained in (a); c) transfecting secondary cells produced in (b)with exogenous DNA encoding erythropoietin and operatively linked to DNAsequences of non-retroviral origin sufficient for expression of theexogenous DNA in transfected secondary cells, thereby producing amixture of secondary cells which includes transfected secondary cellswhich express the exogenous DNA encoding erythropoietin; d) culturingthe product of (c) under conditions appropriate for propagation oftransfected secondary cells which express the exogenous DNA encodingerythropoietin, thereby producing a heterogenous cell strain oftransfected secondary cells of mammalian origin which express theexogenous DNA encoding erythropoietin; and e) introducing transfectedsecondary cells produced in (d) into a mammal of the same species as themammal from which the primary cells were obtained in sufficient numberto provide erythropoietin to the mammal.
 8. The method of claim 7wherein the primary cells are selected from the group consisting of:fibroblasts, keratinocytes, epithelial cells, endothelial cells, glialcells, neural cells, lymphocytes, bone marrow cells, hepatocytes, andprecursors thereof.
 9. A method of providing erythropoietin to a mammal,comprising introducing into the mammal a barrier device containing:a)transfected primary cells that are of the same species as the mammal andthat express exogenous DNA encoding erythropoietin, b) transfectedsecondary cells that are of the same species as the mammal and thatexpress exogenous DNA encoding erythropoietin, c) or both a) andb),wherein the barrier device is made of a material which permitspassage of erythropoietin into the circulation or tissues of the mammaland prevents contact between the immune system of the mammal and thetransfected cells contained within the barrier device to a sufficientextent to prevent a deleterious immune response by the mammal.